Evidence for a role of human organic anion transporters in the muscular side effects of HMG-CoA reductase inhibitors
Introduction
The excretion of numerous organic anions, including endogenous metabolites, drugs, and xenobiotics, is an important physiological function of the kidney. Recently, cDNAs encoding the human organic anion transporters (human OATs) have been successively cloned, including human OAT1 Reid et al., 1998, Hosoyamada et al., 1999, human OAT2 (Enomoto et al., 2002), human OAT3 (Cha et al., 2001), and human OAT4 (Cha et al., 2000). We have been elucidating the localization and functional roles of human OATs mainly in the kidney.
3-Hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase is the rate-limiting step in cholesterol biosynthesis from acetate. The inhibition of this enzyme reduces cytoplasmic cholesterol levels in hepatocytes, which respond by increasing the synthesis of low-density lipoprotein (LDL) receptors that are expressed on hepatocyte surface membranes. This in turn increases hepatic LDL uptake from the plasma, reducing the plasma LDL concentration (Ritter et al., 1999). Thus, HMG-CoA reductase inhibitors have been used to lower plasma total and LOL levels in patients with hypercholesterolemia, which prevents the progression of atherosclerosis.
It has been recently shown that pravastatin, a HMG-CoA reductase inhibitor that possesses anionic moieties, is transported by rat OAT1 and rat OAT3 (Hasegawa et al., 2002). In addition to the kidney, mRNAs from human OATs were shown to be expressed in various organs including the brain, liver, skeletal muscles, and placenta Hosoyamada et al., 1999, Cha et al., 2000, Cha et al., 2001. In the human skeletal muscles, HMG-CoA reductase inhibitors, including pravastatin, fluvastatin, and simvastatin (Fig. 1), were shown to induce various forms of skeletal muscle abnormalities ranging from mild myopathy to myositis, and occasionally rhabdomyolysis and even death (Evans and Rees, 2002). These lines of evidence raise the possibility that human OAT1 and human OAT3 localized in the cytoplasmic membrane of the skeletal muscles mediate the uptake of HMG-CoA reductase inhibitors into the skeletal muscles and the induction of skeletal muscle abnormalities. In order to elucidate this hypothesis, firstly, we performed an immunohistochermical analysis of human OAT1 and human OAT3 in the human skeletal muscles. Secondly, we examined whether human OAT1 and human OAT3 mediate the transport of pravastatin. Thirdly, we elucidated the interactions of human OAT1 and human OAT3 with HMG-CoA reductase inhibitors, including fluvastatin and simvastatin. The activities of human OATs were measured using mouse cell lines from renal proximal tubules stably expressing human OATs.
Section snippets
Materials
Adult human normal skeletal muscle tissue slides were purchased from Bio-chain (Hayward, CA, USA). The human skeletal muscle tissues were from a 26-year-old male. Other materials used included fetal bovine serum, trypsin, and geneticin from Invitrogen (Carlsbad, CA); recombinant epidermal growth factor from Wakunaga (Hiroshima, Japan); insulin from Shimizu (Shizuoka, Japan); RITC 80-7 culture medium from Iwaki (Tokyo, Japan); and simvastatin from Wako (Tokyo, Japan). [14C]pravastatin and
Immunolocalization of human OAT1 and human OAT3 in human skeletal muscles
As shown in Fig. 2, human OAT1 (A) and human OAT3 (B) were stained in the cytoplasmic membrane of the skeletal muscles, but the muscle fibers were not stained. The negative control exhibited no immunoreactivity (C).
Pravastatin uptake mediated by human OAT1 and human OAT3
We examined whether human OAT1 and human OAT3 mediate the uptake of pravastatin. As shown in Fig. 3, human OAT3, but not human OAT1, exhibited significantly higher uptake activities of pravastatin than mock cells (n=8, *P<0.001 vs. mock).
Effects of fluvastatin and simvastatin on organic anion uptake mediated by human OAT1 and human OAT3
We examined the inhibitory effects of various
Discussion
Among adverse drug reactions by HMG-CoA reductase inhibitors, skeletal muscle abnormalities and hepatotoxicity are the most clinically important (Omar et al., 2001). Increases of creatine kinase concentrations to more than three times the upper limit of normal have been reported in 3–5% of patients Omar et al., 2001, Stalenhoef et al., 1989, Ziegler and Drouin, 1990. Skeletal muscle abnormalities can range from benign myalgia to myopathy, which is defined as a 10-fold elevation of the creatine
Acknowledgments
This study was supported, in part, by grants-in-aid from the Ministry of Education, Culture, Sports, Science, and Technology (nos. 11671048, 11694310, 13671128, and 15590858), the Science Research Promotion Fund of the Japan Private School Promotion Foundation, and the fund for Research on Health Sciences Focusing on Drug Innovation from the Japan Health Sciences Foundation.
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